The invention relates to the microbial synthesis of polyhydroxyalkanoic acids [PHA(B)] from substrates which have potential environmental toxicity and must therefore be detoxified and which, if capable of being utilized as a source of carbon and energy by microorganisms for growth and propagation, exhibit the phenomenon of substrate inhibition.
More than 150 species of bacteria from more than 50 genera are known to form polyhydroxybutyric acid (PHB) [Steinb{overscore (u)}chel, A. (1996) Biotechnology (ed. H.-J. Rehm et al.) Vol. 6, Products of Primary Metabolism (ed. M. Roehr), VCH Weinheim, 403-464]. The formation of PHB is not associated with a particular substrate or a particular type of nutrition [Babel, W. (1992) FEMS Microbiology Rev. 103, 141-148] and may proceed starting from CO2 in a chemolitho-autotrophic fashion and also, from reduced organic carbon compounds. Hydrocarbons such as methane, alcohols such as methanol, and acids such as acetic acid and lactic acid are also possible, as well as sugars. More than 50 different substances have been tested.
Processes for the bacterial synthesis of PHB are well-known [Lee, S. Y. (1996). Biotechnol. Bioeng. 49, 1-14]. Substrates most frequently used are carbohydrates as so-called renewable raw materials. Being readily available and relatively favorable in cost, attempts to render methanol and methane commercially acceptable for the synthesis of polyhydroxyalkanoic acids have been made for quite some time. Nevertheless, the price of the product still is high, so that PHB cannot compete with polypropylene and polyethylene plastic materials which are produced on a huge scale and have similar properties, but no (microbiological degradability, and therefore represent a problem when disposed in the form of waste. Since the price of the product is decisively determined by the price of the raw material [Babel, W. (1997) BIOWORLD 4/97, 16-20], more cost-effective carbon sources for the PHB synthesis have to be developed.
Most of the processes for producing PHB described in the literature accumulate PHB(A) as a result of imbalances in the nutrient supply, and in batch cultivation in the stationary phase. As a result of a developing and continuing deficiency e.g. in nitrogen, oxygen or phosphorus (in the form of phosphate), the propagation rate is reduced, and PHB formation is initiated.
Typically, the formation of PHB proceeds independently of growth, so that rendering a process for the synthesis thereof a continuous one is not easy. EP 0,149,744 A1 describes a continuous process. Certainly, this process is based on a special feature of Alcaligenes latus which, in case of complete supply with nutrients optimum for growth and under non-limited conditions of growth, is capable of synthesizing PHB from sugars. This process enables high PHB accumulation either by steady, periodic feeding of substrate (fed-batch regimen), or by a continuous procedure wherein the culture is supplied with a constant flow of fresh nutrient solution and, on the other hand, an aliquot amount of culture medium containing biomass is removed from the fermenter.
Similarly, Methylobacterium rhodesianum must have a specific metabolic/regulatory disposition which can be utilized for continuous PHB production [Ackermann, J.-U., Babel, W. (1997) Appl. Microbiol. Biotechnol. 47, 144-149].
As in the case of A. latus, sugar (in addition to other substrates) is the raw material for PHB synthesis in M. rhodesianum as well.
It has now been found that, as an alternative, PHB can also be produced from substrates having a potential environmental toxicity, and a process regimen has been developed enabling the synthesis of PHB using waste products from the chemical industry and agriculture. Thus, the process according to the invention utilizes cost-effective sources of carbon such as phenol or benzoates, enabling disposal of hazardous substances with simultaneous synthesis of useful materials.
According to the invention, substrates of potential environmental toxicity are employed which feature the phenomenon of substrate inhibition and thus, in the conventional meaning, are unsuitable for the synthesis of over-flow metabolism products, including PHB as well. They are neither suitable in batch operation nor in single-step chemostatic processes limited to carbon substrates, because such conditions which actually prevent growth and propagation and favor PHB synthesis are not realized. Such substrates are aromatic compounds, including phenols, benzoic acid and benzaldehyde. The latter are well-known for their bactericide (bacteriostatic) effect and frequently represent significant components in industrial waste waters.
According to the invention, a process has been developed which can be used to produce PHB from substrates which, when present in excess, exhibit growth inhibition in that appropriate microorganisms utilizing these substrates are chemostatically propagated in such a way that the heat production relative to the substrate flow rate reaches a maximum. Cell growth is monitored calorimetrically, and the maximum heat production corresponding to a maximum PHB content in the biomass is controlled via the substrate flow rate. PHB formation is initiated and controlled by increasing the substrate flow rate at a small volume change.
In a particularly preferred embodiment of the invention, Variovorax paradoxus JMP 116 (DSM No. 4065) is propagated at benzoate flow rates between 0.3 to 1.0 g/lh at rates between 0.07 to 0.4 hxe2x88x921. In another preferred embodiment, Ralstonia eutropha JMP 134 (DSM No. 4058) is propagated at phenol flow rates between 0.3 to 0.6 g/lh at rates between 0.05 to 0.2 hxe2x88x921. It is also preferred to propagate Ralstonia eutropha JMP 134 at benzoate flow rates between 0.25 to 0.7 g/lh at rates between 0.04 to 0.21 hxe2x88x921. The strains that are used are generally available from culture collections.
In a particularly preferred embodiment of the invention, Variovorax paradoxus JMP 116 is propagated at benzoate flow rates between 0.3 to 1.0 g/lh at rates between 0.07 to 0.4 hxe2x88x921. In another preferred embodiment, Ralstonia eutropha JMP 134 is propagated at phenol flow rates between 0.3 to 0.6 g/lh at rates between 0.05 to 0.2 hxe2x88x921. It is also preferred to propagate Ralstonia eutropha JMP 134 at benzoate flow rates between 0.25 to 0.7 g/lh at rates between 0.04 to 0.21 hxe2x88x921. The strains that are used are generally available from culture collections.
According to the invention, a constant amount of heat is withdrawn from the fermenter through a helical heat exchanger in order to determine the heat production in a calorimetric mode. To this end, the mass flow of coolant through the heat exchanger and the temperature difference between inlet and outlet are maintained constant, an electrical heater being controlled in such a way that the reactor temperature remains constant. The difference between the current electric heating power and the one prior to inoculation corresponds to the heat production of the microorganisms.